Antibacterial and antioxidant effect of ethanol extracts of Terminalia chebula on Streptococcus mutans

Abstract

Objective: Dental caries is a high prevalent chronic bacterial infectious disease caused by plaque, a bacterial colony deposited on tooth surfaces and gum tissues. Streptococcus mutans is a primary cariogenic bacterium commonly found in the human oral cavity. Oral hygiene products containing antibacterial ingredients can be helpful in caries management. In this study, we investigated the anticaries mechanism of the ethanol extract of Terminalia chebula (EETC) on S. mutans and suggest its possible application as a functional ingredients for oral hygiene products.

Materials and methods: The EETC was prepared from the Terminalia chebula fruit. Disk diffusion, minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and colony forming unit (CFU) were analyzed to observe the antibacterial activity of EETC. The glucan formation was measured using the filtrate of bacterial culture medium and sucrose. Gene expression was analyzed using RT-PCR. Cytotoxicity was analyzed using the MTT assay. The radical-scavenging activities of DPPH and ABTS were also tested to verify the antioxidant activity of EETC.

Results: The antibacterial activity of the EETC was explored through a disc diffusion analysis and CFU measurement. EETC treatment decreased insoluble glucan formation and gene expression of glycosyltransferase B (gtf B), glycosyltransferase C (gtf C), glycosyltransferase D (gtf D), and fructosyltransferase (ftf). The MIC and MBC of EETC on S. mutans were not cytotoxic to gingival fibroblasts. In addition, we observed DPPH and ABTS-radical scavenging activities of EETC.

Conclusions: These results indicate that the antibacterial and antioxidant effects of EETC may contribute to oral hygiene products for dental caries management.

Keywords: Streptococcus mutans; dental caries; ethanol extract of Terminalia chebula; plaque.

FIGURE 1

Antibacterial effect of EETC on S. mutans growth. S. mutans (1 × 10⁴ CFU) were cultured on an BHI agar plate with a paper disk containing the EETC. The diameter of the bacterial growth inhibition zone was calculated in millimeters (mm)

FIGURE 2

Inhibitory effect of EETC on glucan formation and gene expression related to insoluble saccharide polymer in S. mutans. (a) The filtrate of S. mutans culture medium and sucrose were mixed and kept for 36 h at 37°C. After centrifugation, the precipitate was sonicated and the absorbance was measured at 550 nm. *p < 0.001 versus reaction with the filtrate from S. mutant culture without EETC. (b) RT‐PCR was performed with primers for glycosyltransferase B (gtf B), glycosyltransferase C (gtf C), glycosyltransferase D (gtf D), and fructosyltransferase (ftf). Gene expression was observed in 2% agarose gel electrophoresis. The result shown is the representative images from several experiments. Images were captured and density was measured using ImageJ program. Relative density was plotted in fold changes as a graph. The values of the individual experiments are expressed as the mean ± standard error of three independent experiments. *p < 0.05, **p < 0.001 versus control

FIGURE 3

Effect of EETC on cytotoxicity. Gingival fibroblasts were cultured with or without EETC and live cells were analyzed using MTT assay. Cell viability is shown as % of control. The values of the individual experiments are expressed as the mean ± standard error of three independent experiments. *p < 0.05 versus control

FIGURE 4

Antioxidant effect of EETC. (a) DPPH radical scavenging by EETC. (b) ABTS‐radical scavenging by EETC. Ascorbic acid was used as reductants control. The values of the individual experiments are expressed as the mean ± standard error of three independent experiments. *p < 0.01, *p < 0.001 versus 1 μg/ml EETC